Purpose of Bridging Project (Abstract), Aims and Significance Four of the key aims of the Large Scale grant are: (i) define the specificity and affinity of selected natural and synthetic carbohydrate ligand (ii) establish the cell types involved in cell communication mediated by CBPs (iii) identify the cell types which express the biologically relevant ligands and (iv) determine how CBP-ligand interactions mediate cell communication. For the Siglec subgroup, several novel murine CD33-related Siglecs have been identified and it is likely that more will be discovered on completion of the mouse genome project. These novel proteins will be an important focus of the Large Scale Grant since little is known of (i) their ligand specificity, (ii) the cell types that express them, (iii) the cell types that express their ligands and (iv) their functions. The known human CD33-related Siglec orthologues are expressed mainly on effector cells of the innate immune system and are therefore likely to be important in modulating immune cell activation. Unlike humans, mice provide an excellent model system to study functional aspects of Siglec biology in vivo, but at the present time, crucial reagents such as recombinant forms of Siglecs and specific antibodies are lacking. The purpose of this project is to produce recombinant Fc-chimeric forms of all murine CD33-related Siglecs and generate high quality affinity-purified sheep IgG and Fab fragments against each one. This work will be done in conjunction with the Protein Expression Core. The recombinant proteins will be provided to consortium participants and to the Protein-Carbohydrate Interaction Core to study the specificity for natural and synthetic carbohydrate ligands. They will also be provided to the Mouse Phenotype Core to determine the expression of Siglec ligands. The antibodies will be provided to the Mouse Phenotype Core and participants of the consortium to identify the cell types that express each Siglec and to study their functions in vivo and in vitro. Specific Aims of Bridging Grant Generate constructs encoding cleavable fusion proteins (novel murine Siglecs fused to human IgG1) Produce stable CHO cell lines secreting high levels of recombinant soluble proteins Purify milligram quantities of cleaved, Fc-depleted recombinant protein and immunise sheep Purify IgG and Fab fragments from immune sheep serum Identify novel mouse Siglecs from genomic databases and repeat the above Significance of Bridging Grant For background information on Siglecs please refer to the Program Summary. The reagents generated within this project will interface extensively with the Cores and Participating Investigators and be important in addressing four of the key aims of the Large Scale Project as outlined below: 1. Define specificity and affinity of selected natural and synthetic carbohydrate ligands Recombinant Siglecs fused to the Fc region of human IgG have been used extensively to study specificity for sialylated glycoconjugates 1-8. The recombinant proteins generated in this project will be used for studi3es of specificity by the Paulson laboratory which has an ongoing program to investigate the specificity of novel Siglecs. In addition, the recombinant proteins will be used by the Protein-Carbohydrate Interaction Core H which will study other aspects of Siglec binding proteins in genetically defined mice. 2. Establish the cell types involved in cell communication mediated by CBPs Identification of the cell types that express the novel murine CD33- related Siglecs will critically depend on generation of specific antibodies that can be used im immunohistochemistry and flow cytometry. Although it is expected that monoclonal antibodies may also become available in the future (thorough a collaboration of the Crocker and Varki laboratories with Pharmingen), the affinity purified sheep Igs have the advantage over monoclonal antibodies of increased sensitivity and the ability to recognize multiple epitopes under different conditions of tissue fixation and sample preparation. Immunohistochemistry will be carried out by the Mouse Phenotype Core.